Selenium is an essential micronutrient that exerts many important health benefits. The nutritional requirement for selenium is likely due to its function in selenoproteins, which contain the element in the form of selenocysteine (Sec), the 21st amino acid. Mammalian selenoproteins perform critical functions in thyroid hormone metabolism, anti-oxidant defense inflammation, and development. The goal of this proposal is to understand the mechanism of selenoprotein synthesis and identify the determinants that control the efficiency of this process. The incorporation of Sec into the growing polypeptide chain requires a translational recoding event in which the UGA stop codon is read as Sec. In eukaryotes, the recoding of UGA as Sec depends on the Sec Insertion Sequence (SECIS) in the 3' untranslated region of the selenoprotein mRNA. The SECIS interacts with SECIS Binding Protein 2 (SBP2) and ribosomal protein L30, which play critical roles in the recoding mechanism. We defined a novel bipartite RNA- binding domain in SBP2 and showed that a naturally occurring point mutation in this domain, which is associated with hypothyroidism in humans, alters the SECIS-binding activity of the protein and selectively affects the expression of a subset of selenoproteins, including those involved in thyroid hormone metabolism. Multiple lines of evidence support the hypothesis that L30 is involved in UGA recoding but how this ribosomal protein functions in Sec incorporation is not known. Our studies suggest that there is a dynamic exchange of SBP2 and L30 on the SECIS and that the two proteins act sequentially during UGA recoding. Such a stepwise assembly mechanism may enhance the efficiency of selenoprotein synthesis by preventing nonproductive interactions and orchestrating a specific series of events. In this project, we propose to: 1) understand the molecular basis for the SBP2:SECIS interaction; 2) test the hypothesis that SBP2 dictates the expression of the selenoproteome in vitro and in vivo; and 3) elucidate the function of L30 in selenoprotein synthesis in mammalian cells. The information and molecular tools generated in this project will provide a strong foundation for future studies linking defects in selenoprotein activity in humans to genetic mutations or polymorphisms in genes that encode components of the Sec incorporation pathway. PUBLIC HEALTH RELEVANCE: Selenium, an essential mineral in the diet, is critical for human health. Low dietary intake of selenium, which occurs in many regions of the world, is associated with an increased risk of disease, including thyroid problems, heart disease, inflammation, arthritis, viral infection, and cancer. The goal of this project is to understand how selenium is incorporated into a small but important group of proteins, which play critical roles in the cell and are likely responsible for the beneficial effects of this essential trace element. )